Detection and quantification of nucleic acid molecules constitutes a fundamental trait in several diagnostic techniques. However, the amount of accessible nucleic acid target molecules in a specific sample is in general low, and direct hybridization with a polynucleotide probe therefore in many cases insufficient for detection. In the case of an infectious organism, the classical solution has been enrichment by culturing, but this is time-consuming and therefore unsuitable for rapid diagnosis. A way to circumvent these problems is to amplify the target molecule. This can be accomplished in one of several ways, for example polymerase chain reaction (PCR, as it is described in U.S. Pat. No. 4,683,195 or EP-A-0 630 971), ligase chain reaction (LCR, EP-A-0 320 308) or NASBA (EP-A-329 822). However, all of these techniques utilize enzymes for amplification of target nucleic acid molecules and, consequently, they suffer from the drawback that compounds in the sample specimen may inhibit these enzymes. Therefore, current protocols based on these techniques often include laborious sample preparation steps such as, for example purification and concentration of DNA. Furthermore, when the target sequence is amplified the risk of contamination becomes significantly increased. Several methods have been described to overcome contamination problems, including 1) digestion with nucleases that specifically degrades nucleic acid molecules that have been produced during previous analysis (EP-A-0 401 037); and 2) special laboratory practice that minimises carry over between new samples and previously analysed material. However, methods for avoiding contamination and inhibition are in general costly and cumbersome.
Another solution for signal amplification would be to employ a method in which the number of signal-producing compounds is increased. Such methods have been described by for example EP-A-0 317 077, WO 95/01365 and WO 95/08000. These techniques utilize multi-valent intermediate probes containing a large amount of identical nucleotide sequences bound covalently to each other and each intermediate probe being capable of hybridizing with up to 50 signal producing secondary probes simultaneously. However, these techniques utilize large molecules as intermediate probes which results in less favorable kinetics.
Thus, it would be highly desirable to have a method for signal amplification which is rapid, simple, sensitive, based on stable compounds and on non-enzymatical reactions.